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STING recruits NLRP3 to the ER and deubiquitinates NLRP3 to activate the inflammasome 1
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Wenbiao Wang1, Dingwen Hu2, Yuqian Feng1, Caifeng Wu1, Aixin Li2, Yingchong Wang2, Keli 3
Cheng2, Mingfu Tian2, Feng Xiao2, Qi Zhang2, Muhammad Adnan Shereen2, Weijie Chen1, Pan 4
Pan1, Pin Wan1, Weiyong Liu2, Fang Liu2, Kailang Wu2, Geng Li1, Yingle Liu1,2, and Jianguo Wu1,2* 5
1Guangzhou Key Laboratory of Virology, Institute of Medical Microbiology, Jinan University, 6
Guangzhou 510632, China. 2State Key Laboratory of Virology, College of Life Sciences, Wuhan 7
University, Wuhan 430072, China. 8
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*Correspondence: Jianguo Wu, State Key Laboratory of Virology and College of Life Sciences, 10
Wuhan University, Wuhan 430072, P.R. China, Tel.: +86-27-68754979, Fax: +86-27-68754592, 11
Email: [email protected] 12
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Running title: STING activates the NLRP3 inflammasome 17
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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
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Abstract 20
21
One of the fundamental reactions of the innate immune responses to pathogen infection is the 22
release of pro-inflammatory cytokines, including IL-1β, processed by the NLRP3 inflammasome. 23
STING is essential for innate immune responses and inflammasome activation. Here we reveal a 24
distinct mechanism by which STING regulates the NLRP3 inflammasome activation, IL-1β 25
secretion, and inflammatory responses in human cell lines, mice primary cells, and mice. 26
Interestingly, upon HSV-1 infection and cytosolic DNA stimulation, STING binds to NLRP3 and 27
promotes the inflammasome activation through two approaches. First, STING recruits NLRP3 and 28
promotes NLRP3 translocation to the endoplasmic reticulum, thereby facilitating the inflammasome 29
formation. Second, STING interacts with NLRP3 and removes K48- and K63-linked 30
polyubiquitination of NLRP3, thereby promoting the inflammasome activation. Collectively, we 31
demonstrate that the cGAS-STING-NLRP3 signaling is essential for host defense against DNA 32
virus infection. 33
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Keywords: Cyclic GMP-AMP synthase (cGAS)/Herpes simplex virus type 1 35
(HSV-1)/Interleukine-1(IL-1)/Polyubiquitination and deubiquitination/The cGAS-STING 36
pathway 37
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Introduction 39
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The innate immune system detecting pathogens through recognition of molecular patterns is a 41
primary host defense strategy to suppress the infections (Akira et al, 2006). Recognition of 42
pathogens stimuli, known as pathogen-associate molecular patterns (PAMPS), is relied on pattern 43
recognition receptors (PRRs). Several families of PRRs have been described, including the Toll-like 44
receptor (TLR) (Takeuchi & Akira, 2010), RIG-I-like receptor (RLR) (Yoneyama et al., 2004), 45
NOD-like receptor (NLR) (Ting et al, 2008), and C-type lectin receptor (CLR) (Hardison & Brown, 46
2012). The NLRs involved in the assembly of large protein complexes referred to as 47
inflammasomes are emerging as a major route by which the innate immune system responds to 48
pathogen infections (Schroder & Tschopp, 2010). One of the fundamental reactions of the innate 49
immunity is the procession and release of pro-inflammatory cytokines, including interleukine-1 50
(IL-1β), a pleiotropic cytokine playing crucial roles in inflammatory responses in addition to 51
instructing immune responses (Dinarello, 2009). The best-characterized inflammasomes is the 52
NLRP3 inflammasome, which consists of three major components: a cytoplasmic sensor NLRP3 53
(NACHT, LRR and PYD domains-containing protein 3), an adaptor ASC (apoptosis-associated 54
speck-like protein with CARD domain), and an interleukin-1β-converting enzyme pro-Caspase-1 55
(cysteinyl aspartate-specific proteinase-1) (Schroder & Tschopp, 2010). NLRP3 and ASC together 56
promote the cleavage of pro-Casp-1 to generate active subunits p20 and p10, which regulate IL-1β 57
maturation (Martinon et al, 2004). 58
The stimulator of interferon genes (STING) has the essential roles in innate immune response 59
against pathogen infections (Ishikawa et al, 2009). Upon binding of cytoplasmic DNA, cyclic 60
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GMP-AMP synthase (cGAS) catalyzes the formation of cyclic guanosine monophosphate-adenosine 61
monophosphate (cGAMP) by binding to STING. STING subsequently co-localizes with TBK1 and 62
IRF3, leading to induction of type I IFNs, and recruits TRAF6 and TBK1 or TRAF3 and IKKα to 63
activate the NF-κB pathway (Abe & Barber, 2014; Wu et al., 2013). In human myeloid cells, 64
STING is involved in cytosolic DNA induced-NLRP3 inflammasome activation (Gaidt et al, 2017), 65
and in mice BMDMs, STING is required for pathogen-induced inflammasome activation and IL-1β 66
secretion (Webster et al., 2017; Swanson et al., 2017). 67
We explored how STING regulates the NLRP3 inflammasome and reveal a distinct mechanism 68
underlying such regulation upon herpes simplex virus type 1 (HSV-1) infection and cytosolic DNA 69
stimulation. HSV-1 causes various mild clinical symptoms, while in immunocompromized and 70
neonates individuals, it can cause herpes simplex encephalitis, which may lead to death or result in 71
some neurological problems (Roizman, 2013). But the detailed mechanisms by which HSV-1 72
regulates the NLRP3 inflammasome are largely unknown. We demonstrate that HSV-1-induced 73
NLRP3 inflammasome activation is dependent on the cGAS-cGAMP-STING pathway. STING 74
recruits and promotes NLRP3 translocation to the endoplasmic reticulum, and binds and removes 75
NLRP3 polyubiquitination, thereby promoting the inflammasome activation. We propose that the 76
cGAS-cGAMP-STING-NLRP3 axis is essential for host defense against DNA virus infection. 77
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Results 79
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STING interacts with NLRP3 to facilitate the inflammasome activation. 81
We initially determined the correlation between STING and NLRP3, and showed that STING and 82
NLRP3 interacted with each other in human embryonic kidney (HEK293T) cells (Fig 1A, B). The 83
NLRP3 inflammasome consists of three major components, NLRP3, ASC, and pro-Casp-1 84
(Schroder & Tschopp, 2010). We explored whether STING interacts with ASC and/or pro-Casp-1, 85
and clearly revealed that STING interacted with NLRP3, but not with ASC or pro-Casp-1 (Fig 1C). 86
NLRP3 protein harbors several prototypic domains, including PYRIN domain (PYD), 87
NACHT-associated domain (NAD), and Leucine rich repeats (LRR) (Ye & Ting, 2008). Next, the 88
domain of NLRP3 involved in the interaction with STING was determined by evaluating the 89
plasmids encoding NLRP3, PYRIN, NACHT, or LRR (Figure 1D) as described previously (Wang et 90
al, 2018). Like NLRP3, NACHT and LRR interacted with STING, but PYRIN failed to interact 91
with STING (Figure 1E), and consistently, STING interacted with NLRP3, NACHT, and LRR (Fig 92
1F, lanes 2, 6 and 8), but not with PYRIN (Fig 1F, lane 4). In another hand, STING comprises five 93
putative transmembrane (TM) regions (Ishikawa & Barber, 2008). The domain of STING required 94
for the interaction with NLRP3 was assessed by analyzing plasmids encoding wild-type (WT) 95
STING and seven truncated proteins (Fig 1G). Like WT STING(1–379aa) (Fig 1H, lane 9), the 96
truncated proteins STING(1–160aa), STING(1–240aa), STING(41–379aa), STING(81–379aa), and 97
STING(111–379aa) interacted strongly with NLRP3 (Fig 1H, lane 2–6), STING(151–379aa) 98
associated weakly with NLRP3 (Fig 1H, lane 7), but STING(211–379aa) failed to interact with 99
NLRP3 (Fig 1H, lane 8), indicating that TM5 (151–160aa) of STING is involved in the interaction 100
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with NLRP3. 101
The role of STING in the regulation of the NLRP3 inflammasome was explored by using a 102
pro-Casp-1 activation and pro-IL-1β cleavage cell system as established previously (Wang et al., 103
2017). In this system (NACI), HEK293T cells were co-transfected with plasmids encoding NLRP3, 104
ASC, pro-Casp1, and pro-IL-1β. In the NACI cells, IL-1β secretion was stimulated by STING(1–105
160aa), STING(1–240aa), STING(41–379aa), or STING (Fig 1I, lane 2–4 and 9), but not by 106
STING(81–379aa), STING(111–379aa), STING(151–379aa) or STING(211–379aa) (Fig 1I, lane 5–107
8), suggesting that TM2 (41–81aa) of STING is required for the induction of IL-1β secretion. 108
Notably, STING promoted the NLRP3-ASC interaction (Fig 1J), an indicator of the inflammasome 109
assembly (Compan et al., 2012) and enhanced NLRP3-mediated ASC oligomerization (Fig 1K), 110
which is critical for inflammasome activation (Shenoy et al., 2012). Moreover, in HEK293T cells 111
(Fig 1L) and HeLa cells (Fig 1M), NLRP3 and STING co-localized and formed large spots in the 112
cytosol (Fig 1L and M), an indication of the NLRP3 inflammasome formation ((Martinon et al., 113
2009). Taken together, STING interacts with NLRP3 through TM5 domain and promotes the 114
assembly and activation of the NLRP3 inflammasome through TM2 domain. 115
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HSV-1 infection promotes the STING-NLRP3 interaction. 117
STING plays a key role in host innate immune response in response to pathogen infections and 118
cytosolic DNA simulation (Ishikawa & Barber, 2008). We evaluated the effects of HSV-1 infection 119
and HSV120 transfection, a biotinylated dsDNA representing the genomes of HSV-1 that efficiently 120
induces STING-dependent type I IFN production as reported previously (Abe et al., 2013) on the 121
STING-NLRP3 interaction. In TPA-differentiated human leukemic monocyte (THP-1) macrophages, 122
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HSV-1 infection facilitated endogenous NLRP3-STING interaction and promoted endogenous 123
STING-NLRP3 interaction (Fig 2A, B). In HEK293T cells and HeLa cells (Fig 2D), the 124
STING-NLRP3 interaction was enhanced upon HSV-1 infection (Fig 2C, D). Similarly, in 125
TPA-differentiated THP-1 macrophages, HEK293T cells, and HeLa cells, the NLRP3-STING 126
interaction was promoted by HSV120 transfection (Fig 2E−G). Moreover, in primary mouse 127
embryo fibroblasts (MEFs), HSV-1 infection and HSV120 transfection facilitated endogenous 128
STING-NLRP3 interaction (Fig 2H). Collectively, HSV-1 infection and HSV120 transfection 129
facilitate the interaction of STING with NLRP3. 130
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HSV-1 infection induces IL-1β expression and secretion. 132
Next, we explored whether HSV-1 infection and HSV120 transfection regulate the NLRP3 133
inflammasome activation. In TPA-differentiated THP-1 macrophages, endogenous IL-1β secretion 134
was induced by Nigericin (a positive control for the inflammasome activation) and HSV-1 (Fig 3A, 135
B). Consistently, IL-1β maturation and Casp-1 cleavage, as well as pro-IL-1β production were 136
activated upon HSV-1 infection (Fig 3C, D). Notably, IL-1β mRNA was not induced by Nigericin 137
but induced upon HSV-1 infection (Fig 3E, F) and HSV-1 ICP27 mRNA was expressed in the 138
infected cells (Fig 3G, H). Similarly, in TPA-differentiated THP-1 macrophages, IL-1β secretion 139
was induced by Nigericin and facilitated by HSV120 (Fig 3I). IL-1β maturation and Casp-1 140
cleavage and pro-IL-1β production were stimulated by Nigericin and promoted by HSV120 (Fig 3J). 141
IFN-β mRNA expression was not induced by Nigericin but activated by HSV120 (Fig 3K), 142
demonstrating that HSV120 is effective in the cells. Moreover, in mice primary MEFs, endogenous 143
IL-1β secretion was induced by ATP (a positive control), promoted upon HSV-1 infection, and 144
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enhanced by HSV120 stimulation (Fig 3L, M). HSV-1 ICP27 mRNA was detected in the cells (Fig 145
3N), suggesting that HSV-1 is replicated. IFN-β mRNA was not induced by ATP but activated by 146
HSV120 in the cells (Fig 3O), demonstrating that HSV120 is effective. Therefore, IL-1β expression 147
and secretion are induced upon DNA virus infection and cytosolic DNA stimulation. 148
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The NLRP3 inflammasome is required for HSV-1-induced IL-1β activation. 150
Accordingly, we determined whether the NLRP3 inflammasome is required for HSV-1 in the 151
induction of IL-1β activation. Initially, the effects of glybenclamide (NLRP3 inhibitor) and 152
Ac-YVAD-cmk (Casp-1 inhibitor) (Wang et al., 2015) on HSV-1-mediated IL-1β activation were 153
evaluated. In TPA-differentiated THP-1 macrophages, IL-1β secretion (Fig 4A, C) as well as IL-1β 154
(p17) cleavage and Casp-1(p20 and p22) maturation (Fig 4B, D) activated by Nigericin and HSV-1 155
were significantly attenuated by glybenclamide (Fig 4A, B) or Ac-YVAD-cmk (Fig 4C, D). HSV-1 156
ICP27 mRNA was expressed in infected cells (Fig 4E, F), indicating that HSV-1 is replicated. 157
In addition, the role of endogenous NLRP3 inflammasome in HSV-1-induced IL-1β activation 158
was assessed in THP-1 cell lines stably expressing negative control shRNA (sh-NC) and shRNAs 159
(sh-NLRP3, sh-ASC and sh-Casp-1) targeting the NLRP3 inflammasome components. Notably, 160
IL-1β secretion (Fig 4G, I) as well as IL-1β (p17) cleavage and Casp-1 (p20 and p22) maturation 161
(Fig 4H, J, top) induced by Nigericin (Fig 4G, H) or HSV-1 (Fig 4I, J) were attenuated by 162
sh-NLRP3, sh-ASC, and sh-Casp-1. The NLRP3, ASC, and pro-Casp-1 proteins were 163
own-regulated by sh-NLRP3, sh-ASC, and sh-Casp-1 stable cells, respectively (Fig 4H, J, bottom), 164
indicating that shRNAs are effective in the cells. HSV-1 ICP27 mRNA was expressed in infected 165
cells (Fig 4K), confirming that HSV-1 is replicated in the cells. 166
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Moreover, the direct role of NLRP3 in the regulation of HSV-1-induced IL-1β secretion was 167
determined in primary MEFs of C57BL/6 WT pregnant mice and NLRP3-/- pregnant mice. NLRP3 168
protein was detected in WT mice primary MEFs, but not in NLRP3-/- mice primary MEFs (Fig 4L), 169
indicating that NLRP3 is knocked out in the null mice. IL-1β secretion was induced by ATP and 170
HSV-1 in WT mice primary MEFs but not in NLRP3-/- mice primary MEFs (Fig 4M). HSV-1 ICP27 171
mRNA was expressed in infected cells, indicating that HSV-1 is replicated in the cells (Fig 4N). 172
Collectively, inhibition, knock-down, and knock-out of the NLRP3 inflammasome components lead 173
to the repression of IL-1β secretion and Casp-1 maturation, therefore the NLRP3 inflammasome is 174
required for HSV-1-induced activation of IL-1β. 175
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STING recruits NLRP3 to the ER to promote the inflammasome formation. 177
STING predominantly resides in the endoplasmic reticulum (ER) to regulate innate immune 178
signaling processes (Ishikawa & Barber, 2008). Here we evaluated whether STING recruits NLRP3 179
to ER and activates the inflammasome. In HeLa cells, NLRP3 alone diffusely distributed in the 180
cytoplasm and STING alone located in the ER (as indicated by the ER marker, ER blue), while 181
NLRP3 and STING together co-localized and distributed in the ER to form specks (Fig 5A), which 182
is an indication of the NLRP3 inflammasome complex formation (Martinon et al., 2009), suggesting 183
that STING recruits NLRP3 to the ER and promotes the inflammasome formation. In addition, 184
NLRP3 diffusely distributed in the cytosol of untreated cells, but formed distinct specks upon 185
HSV-1 infection or HSV120 transfection (Fig 5B). Moreover, NLRP3 diffusely distributed in the 186
cytosol of untreated cells, while upon HSV-1 infection or HSV120 transfection, NLRP3 forms 187
distinct specks in the ER as indicated by Calnexin (ER protein) (Fig 5C) and ER blue (ER marker) 188
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(Fig 5D). Notably, in transfected HeLa cells, NLRP3, STING, and Calnexin were detected in whole 189
cell lysate (WCL) and purified ER fraction, and interestingly, NLRP3 abundance was enhanced by 190
STING in purified ER fraction (Fig 5E). Similarly, in mock-infected THP-1 macrophages and mice 191
primary MEFs, NLRP3, STING, and Calnexin were detected in WCL and purified ER, and NLRP3 192
abundance was increased in the ER upon HSV-1 infection (Fig 5F, G). Therefore, STING, HSV-1, 193
and HSV120 facilitate the NLRP3 inflammasome formation in the ER. 194
Moreover, the effect of endogenous STING on NLRP3 translocation to the ER was further 195
determined by using shRNA targeting STING (sh-STING). Hela cells stably expressing sh-NC or 196
sh-STING were generated, and then transfected with Flag-NLRP3 and infected with HSV-1 or 197
transfected with HSV120. In the absence of sh-STING, NLRP3 diffusely distributed in the cytosol 198
of untreated cells and formed distinct specks in the ER, as indicated by Calnexin and ER Blue (Fig 199
5H, I, top), upon HSV-1 infection or HSV120-transfection, however, in the presence of sh-STING, 200
NLRP3 failed to form specks upon HSV-1 infection or HSV120 transfection (Fig 5H, I, bottom), 201
indicating that STING knock-down leads to the repression of HSV-1-induced formation of the 202
NLRP3 inflammasome. In addition, NLRP3, Calnexin, and STING were detected in WCL and 203
purified ER fraction of Hela cells (Fig 5J), THP-1 cells (Fig 5K) and mice primary MEFs (Fig 5L), 204
and notably, NLRP3 level was higher in purified ER fraction upon HSV-1 infection in sh-NC stable 205
cells (Fig 5J–L, lane 6 vs. 5) as compared with sh-STING stable cells (Fig 5J–L, lane 8 vs. 7), 206
suggesting that STING knock-down results in the attenuation of NLRP3 translocation to the ER 207
upon HSV-1 infection. We also confirmed that STING abundance was down-regulated by 208
sh-STING (Fig 5J–L). Collectively, STING recruits NLRP3 to the ER and promotes the NLRP3 209
inflammasome formation upon HSV-1 infection and cytosolic DNA stimulation. 210
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211
STING deubiquitinates NLRP3 to activate the NLRP3 inflammasome. 212
The deubiquitination of NLRP3 is required for the NLRP3 inflammasome activation (Juliana et al., 213
2012). We next investigated whether STING plays a role in the deubiquitination of NLRP3, thereby 214
facilitating the inflammasome activation. Interestingly, NLRP3 polyubiquitination catalyzed by 215
HA-UB, HA-UB(K48R), or HA-Ub(K63R) was repressed by STING (Fig 6A, B). We also revealed 216
that NLRP3 polyubiquitination catalyzed by HA-UB, HA-UB(K48O) (ubiquitin mutant that only 217
retains a single lysine residue), or HA-UB(K63O) (ubiquitin mutant that only retains a single lysine 218
residue) was suppressed by STING (Fig 6C). These results reveal that STING removes K48- and 219
K63-linked polyubiquitination of NLRP3. 220
In addition, we examined whether HSV-1 infection and HSV120 transfection regulate the 221
deubiquitination of NLRP3. Notably, NLRP3 polyubiquitination catalyzed by HA-UB was 222
attenuated upon HSV-1 infection (Fig 6D) or by HSV120 transfection (Fig 6E). In THP-1 223
differentiated macrophages, UB-catalyzed (Fig 6F, top), K48-linked (Fig 6F, middle), and 224
K63-linked (Fig 6F, bottom) polyubiquitination of endogenous NLRP3 was repressed upon HSV-1 225
infection; and UB-catalyzed polyubiquitinion of endogenous NLRP3 was repressed by HSV120 226
(Fig 6G). Moreover, in mice primary MEFs, UB-catalyzed ployubiquitination of endogenous 227
NLRP3 was attenuated upon HSV-1 infection (Fig 6H). Therefore, HSV-1 infection and HSV120 228
stimulation promote the deubiquitination of endogenous NLRP3. Moreover, upon HSV-1 infection, 229
the ployubiquitination of endogenous NLRP3 was attenuated in the presence of sh-NC but relatively 230
unaffected in the presence of sh-STING (Fig 6I), indicating that STING knock-down leads to 231
repression of NLRP3 deubiquitination. Taken together, STING removes K48- and K63-linked 232
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polyubiquitination of NLRP3 to promote the inflammasome activation upon HSV-1 infection and 233
cytosolic DNA stimulation. 234
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STING is required for the NLRP3 inflammasome activation upon DNA virus infection. 236
Since STING recruits NLRP3 to the ER and removes NLRP3 deubiquitination upon HSV-1 237
infection, we speculated that STING may play a role in HSV-1-induced NLRP3 inflammasome 238
activation. The effect of STING knock-down on HSV-1-induced NLRP3 inflammasome activation 239
was initially examined in THP-1 cells stably expressing sh-STING. Endogenous IL-1β secretion as 240
well as IL-1β (p17) cleavage and Casp-1 (p20 and p22) maturation induced by HSV-1 were 241
significantly attenuated by sh-STING (Fig 7A, B). HSV-1 ICP27 mRNA was detected in HSV-1 242
infection cells (Fig 7C), indicating that HSV-1 is replicated. Additionally, endogenous IL-1β 243
secretion, IL-1β (p17) maturation, and Casp-1 (p20 and p22) cleavage induced by DNA90, HSV120, 244
or HSV-1 were suppressed by sh-STING (Fig 7D, E). Thus, STING knock-down leads to the 245
suppression of IL-1β secretion and Casp-1 maturation upon DNA virus infection and cytosolic DNA 246
stimulation. 247
Accordingly, we determine whether STING plays a specific role in the NLRP3 inflammasome 248
activation mediated by DNA virus. THP-1 cells stably expressing sh-STING were differentiated to 249
macrophages, and then treated with Nigericin or infected with HSV-1. Notably, sh-STING 250
significantly attenuated endogenous IL-1β secretion as well as L-1β (p17) maturation and Casp-1 251
(p20 and p22) cleavage induced upon HSV-1 infection, but had no effect on IL-1β secretion or 252
IL-1β (p17) maturation and Casp-1 (p20 and p22) cleavage induced by Nigericin stimulation (Fig 7F, 253
G). HSV-1 ICP27 mRNA was detected in HSV-1 infection cells (Fig 7H), indicating that HSV-1 is 254
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replicated in the cells. Moreover, we explored whether STING plays roles in the NLRP3 255
inflammasome activation induced by RNA viruses. THP-1 differentiated macrophages stably 256
expressing sh-STING were infected with RNA viruses, Sendai virus (SeV) and Zika virus (ZIKV), 257
or with HSV-1. Interestingly, endogenous IL-1β secretion as well as IL-1β (p17) maturation and 258
Casp-1 (p20 and p22) cleavage were induced upon the infections of the three viruses, however, 259
HSV-1-mediated induction was attenuated by sh-STING, but SeV- or ZIKV-mediated inductions 260
were not affected by sh-STING (Fig 7I, J). SeV P mRNA, ZIKV mRNA, and HSV-1 ICP27 mRNA 261
were detected in infected cells, respectively (Fig 7K–M). Taken together, STING plays specific 262
roles in the NLRP3 inflammasome activation upon DNA virus infection or cytosolic DNA 263
stimulation, but has no effect on the inflammasome activation induced by RNA virus infection or 264
Nigericin stimulation. 265
266
NLRP3 is critical for host defense against HSV-1 infection in mice. 267
To gain insights into the biological function of NLRP3 in vivo, we analyzed C57BL/6 NLRP3+/+ 268
WT mice and C57BL/6 NLRP3-/- deficiency mice. Notably, HSV-1-infected NLRP3-/- mice began to 269
die at 5 days post-infection and all infected mice died at 7 days post-infection, while infected WT 270
mice began to die at 7 days post-infection and 30% WT mice was survival after 11 days 271
post-infection (Fig 8A). The body weights of infected NLRP3-/- mice decreased continuously until 272
died, while the body weights of infected WT mice gradually decreased until 7 days post-infection 273
and then gradually increased (Fig 8B). Thus, NLRP3 deficiency mice are more susceptibility to 274
HSV-1 infection and exhibit early onset of death upon the infection. 275
Notably, in the mice blood, IL-1β secretion was induced upon HSV-1 infection in WT mice, 276
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whereas it was not induced in NLRP3-/- mice (Fig 8C), IL-1β mRNA level was higher in WT mice 277
as compared with NLRP3-/- mice (Fig 8D), IL-6 mRNA and TNF-α mRNA were no significant 278
difference between WT and NLRP3-/- mice (Fig 8E, F), and HSV-1 UL30 mRNA was expressed in 279
infected WT and NLRP3-/- mice (Fig 8G). These results indicate that NLRP3 deficiency leads to the 280
repression of IL-1β expression and secretion in mice. Interestingly, in HSV-1 infected mice lung and 281
brain, IL-1β mRNA and IL-6 mRNA were significantly higher in WT mice as compared with 282
NLRP3-/- mice (Fig 8H, I, K and L), however, the viral titers were much lower in WT mice as 283
compared with NLRP3-/- mice (Fig 8J, M), suggesting that NLRP3 deficiency results in the 284
attenuation of IL-1β expression and the promotion of HSV-1 replication in mice lung and brain. 285
Moreover, Hematoxylin and Eosin (H&E) staining showed that more infiltrated neutrophils and 286
mononuclear cells were detected in the lung and brain of infected WT mice as compared with 287
NLRP3-/- mice (Fig 8N), and immunohistochemistry (IHC) analysis revealed that IL-1β protein 288
level was higher in the lung and brain of infected WT mice as compared with NLRP3-/- mice (Fig 289
8O), revealing that NLRP3 deficiency mice are more susceptibility to HSV-1 infection and elicit 290
weak inflammatory responses. Collectively, we propose that NLRP3 is essential for host defense 291
against HSV-1 infection by facilitating IL-1β activation (Fig 9). 292
293
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Discussion 294
295
This study reveals a distinct mechanism by which the cGAS-STING-NLRP3 pathway promotes the 296
NLRP3 inflammasome activation and IL-1β secretion upon DNA virus infection and cytosolic DNA 297
stimulation. The cGAS-STING pathway mediates immune defense against infection of 298
DNA-containing pathogens and detects tumor-derived DNA and generates intrinsic antitumor 299
immunity (Chen et al., 2016; Wu et al., 2013). More recent studies reported that in human 300
monocytes, the cGAS-STING pathway is essential for cytosolic DNA induced-NLRP3 301
inflammasome (Gaidt et al., 2017) and in mice BMDMs, the cGAS-STING pathway is required for 302
Chlamydia trachomatis-induced inflammasome activation and IL-1β secretion (Webster et al., 2017; 303
Swanson et al., 2017). Our results are consistent with the reports and further support that 304
cGAS-STING pathway is essential not only for cytosolic DNA induced- or Chlamydia 305
trachomatis-induced NLRP3 inflammasome activation, but also for DNA virus-induced NLRP3 306
inflammasome activation. This study also further reveals that the cGAS-STING pathway is required 307
for the NLRP3 inflammasome activation not only in human monocytes and mice BMDMs, but also 308
in human embryonic kidney cells (HEK293T), Hela cells, human leukemic monocytes/macrophages 309
(THP-1), and mice primary mouse embryo fibroblasts (MEFs). More interestingly, our results reveal 310
a distinct mechanism underlying STING-mediated NLRP3 inflammasome activation, and 311
demonstrate for the first time that STING binds to NLRP3 and promotes the inflammasome 312
activation through two approaches. First, STING binds to and recruits NLRP3 to the ER to promote 313
the formation of the NLRP3 inflammasome. Second, STING interacts with NLRP3 and removes 314
K48- and K63-linked polyubiquitination of NLRP3 to induce the activation of the NLRP3 315
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16
inflammasome. Notably, upon HSV-1 infection and HSV120 stimulation, STING binds to NLRP3, 316
promotes the NLRP3-ASC interaction (an indicator of inflammasome complex assembly) (Compan 317
et al., 2012), facilitates NLRP3-mediated ASC oligomerization (a critical step for inflammasome 318
activation) (Shenoy et al., 2012), enhances NLRP3 to form specks (an indicator of inflammasome 319
activation) (Martinon et al, 2009), and enhances IL-1β secretion (a fundamental reaction of the 320
inflammatory responses) (Dinarello, 2009). Collectively, the cGAS-STING-NLRP3 pathway plays 321
key roles in the NLRP3 inflammasome activation and IL-1β secretion upon DNA virus infection 322
and cytosolic DNA stimulation. 323
Notably, STING knock-down attenuates the NLRP3 inflammasome activation mediated upon 324
DNA virus infection or cytosolic DNA stimulation, but has no effect on the NLRP3 inflammasome 325
activation induced by RNA virus infection or Nigericin induction. Many RNA viruses induce the 326
NLRP3 inflammasome activation, including influenza A virus (IAV) (Kanneganti et al., 2006), 327
Vesicular mastitis virus (VSV) and Encephalomyocarditis virus (EMCV) (Rajan et al., 2011), 328
Measles virus (MV) (Komune et al., 2011), West Nile virus (WNV) (Ramos et al., 2012), Rabies 329
virus (RV) (Lawrence et al., 2013), Hepatitis C virus (HCV) (Negash et al., 2013), Dengue virus 330
(DENV) (Hottz et al., 2013), Enterovirus 71 (EV71) (Wang et al., 2017) and Zika virus (ZIKV) 331
(Wang et al., 2018). Some DNA viruses also regulate the NLRP3 inflammasome activation, such as 332
Adenovirus (AdV) (Muruve et al., 2008) and HSV-1 (Johnson et al., 2013). HSV-1 VP22 inhibits 333
AIM2-denpendent inflammasome activation so that HSV-1 infection of macrophages-induced 334
inflammasome activation is AIM2-independent (Maruzuru et al., 2018). Here, we demonstrate that 335
the cGAS-STING-NLRP3 pathway is required for HSV-1-induced NLRP3 inflammasome 336
activation and critical for host defense against DNA virus infection. 337
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The mechanisms of NLRP3 inflammasome activation have been intensely studied. The 338
mitochondria-associated adaptor protein (MAVS) promotes NLRP3 mitochondrial localization and 339
the inflammasome activation (Subramanian et al., 2013). PtdIns4P mediates the NLRP3 340
inflammasome activation in trans-Golgi network (TGN) (Chen & Chen, 2018). NLRP3 associates 341
with SCAP-SREBP2 to form a ternary complex that translocates to the Golgi apparatus for optimal 342
inflammasome assembly (Guo et al., 2018). Here we find that STING promotes NLRP3 343
translocation to the ER and facilitates the inflammasome activation. Moreover, post-translational 344
modifications of NLRP3 are critical for its activation, including phosphorylation (Song & Li, 2018), 345
SUMOylation (Barry et al., 2018), and ubiquitination (Py et al., 2013). MARCH7 and TRIM31 346
facilitate NLRP3 ubiquitination and proteasomal degradation (Song et al., 2016; Yan et al., 2015). 347
Pellino2 promotes K63-linked ubiquitination of NLRP3 as part of the priming phase (Humphries et 348
al., 2018). Interestingly, we demonstrate that STING removes K48- and K63-linked ubiquitination 349
of NLRP3 to promote the inflammasome activation, and reveal that HSV-1 infection induces 350
STING-mediated deubiquitination of NLRP3. 351
Moreover, NLRP3 is related to many human diseases. Fibrillar amyloid-β peptide, the major 352
component of Alzheimer’s disease brain plaques, facilitates the NLRP3 inflammasome activation 353
(Halle et al., 2008). Monosodium urate (MSU) crystals induce the autoinflammatory disease gout 354
and activate the NLRP3 inflammasome (Martinon et al., 2006). NLRP3, IL-1β, reactive oxygen 355
species (ROS), and TXNIP are implicated in the type 2 diabetes mellitus (T2DM) pathogenesis 356
(Schroder et al., 2010). Our study gains insights into the biological function of the 357
cGAS-STING-NLRP3 pathway in host defense against HSV-1 infection in mice. NLRP3 deficiency 358
mice are more susceptibility to HSV-1 infection, exhibit early onset of death upon infection, 359
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represses IL-1β secretion, and elicits robust inflammatory responses in the tissues. Collectively, 360
these results demonstrate that NLRP3 is essential for host defense against HSV-1 infection by 361
inducting IL-1β expression and secretion. 362
In conclusion, we reveal a distinct mechanism underlying the regulation of the NLRP3 363
inflammasome activation upon DNA virus infection. In this model, STING (the central molecule of 364
the antiviral and inflammatory immune pathways) interacts with NLRP3 (the key component of the 365
inflammasomes), removes NLRP3 polyubiquitination, recruits NLRP3 to the ER, and facilitates the 366
NLRP3 inflammasome activation, thereby inducing IL-1β secretion upon DNA virus infection and 367
cytosolic DNA stimulation. 368
369
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Materials and Methods 370
371
Animal study 372
C57BL/6 WT mice were purchased from Hubei Research Center of Laboratory Animals 373
(Wuhan, Hubei, China). C57BL/6 NLRP3-/- mice were kindly provided by Dr. Di Wang of Zhejiang 374
University School of Medicine, China. 375
The primary mouse embryo fibroblasts (MEFs) were prepared from pregnancy mice of 376
C57BL/6 WT mice and C57BL/6 NLRP3-/- mice in E14 and cultured in Dulbecco modified Eagle 377
medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS). 378
379
Ethics statement. 380
All animal studies were performed in accordance with the principles described by the Animal 381
Welfare Act and the National Institutes of Health Guidelines for the care and use of laboratory 382
animals in biomedical research. All procedures involving mice and experimental protocols were 383
approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Life 384
Sciences, Wuhan University. 385
386
Cell lines and cultures 387
African green monkey kidney epithelial (Vero) cells, human cervical carcinoma (Hela) cells, 388
and human embryonic kidney 293T (HEK 293T) cells were purchased from American Type Culture 389
Collection (ATCC) (Manassas, VA, USA). Human acute monocytic leukemia (THP-1) cells were 390
gift from Dr. Jun Cui of State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen 391
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20
University, Guangzhou 510275, China. THP-1 cells were cultured in RPMI 1640 medium 392
supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 393
μg/ml streptomycin sulfate. Vero, Hela and HEK293T cells were cultured in Dulbecco modified 394
Eagle medium (DMEM) purchased from Gibco (Grand Island, NY, USA) supplemented with 10% 395
FBS, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate. Vero, Hela, HEK293T and THP-1 396
cells were maintained in an incubator at 37°C in a humidified atmosphere of 5% CO2. 397
398
Reagents 399
Lipopolysaccharide (LPS), adenosine triphosphate (ATP), Endoplasmic Reticulum Isolation 400
Kit (ER0100), phorbol-12-myristate-13-acetate (TPA) and dansylsarcosine piperidinium salt (DSS) 401
were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640 and Dulbecco modified 402
Eagle medium (DMEM) were obtained from Gibco (Grand Island, NY, USA). Nigericin and 403
Ac-YVAD-cmk were obtained from InvivoGene Biotech Co., Ltd. (San Diego, CA, USA). Antibody 404
against Flag (F3165), HA (H6908) and monoclonal mouse anti-GAPDH (G9295) were purchased 405
from Sigma (St. Louis, MO, USA). Monoclonal rabbit anti-NLRP3 (D2P5E), Ubiquitin mouse mAb 406
(P4D1), monoclonal rabbit anti-K63-linkage Specific Polyubiquitin (D7A11), monoclonal rabbit 407
anti-K48-linkage Specific Polyubiquitin (D9D5), monoclonal rabbit anti-STING(D2P2F), 408
monoclonal rabbit anti-calnexin(C5C9), monoclonal rabbit anti-IL-1β (D3U3E), IL-1β mouse mAb 409
(3A6) and monoclonal rabbit anti-Caspase-1 (catalog no. 2225) were purchased from Cell Signaling 410
Technology (Beverly, MA, USA). Monoclonal mouse anti-ASC (sc-271054) and polyclonal rabbit 411
anti-IL-1β (sc-7884) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 412
Monoclonal mouse anti-NLRP3 (AG-20B-0014-C100) was purchased from Adipogen to detection 413
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endogenous NLRP3 in THP1 cells and primary MEFs. Lipofectamine 2000, ER-TrackerTM 414
Blue-White DPX (E12353), normal rabbit IgG and normal mouse IgG were purchased from 415
Invitrogen Corporation (Carlsbad, CA, USA). 416
417
Viruses 418
Herpes simplex virus 1 (HSV-1) strain and Sendai virus (SeV) strain were gifts from Dr. Bo 419
Zhong of Wuhan University. Zika virus (ZIKV) isolate z16006 (GenBank accession number 420
KU955589.1) was used in this study. Vero cells were maintained at 37°C in DMEM (GIBCO) 421
supplemented with 10% heat-inactivated FBS with penicillin/streptomycin (GIBCO) (Grand Island, 422
NY, USA). HSV-1 stocks were propagated in Vero cells for 36 h at 0.03 MOI. The infected cells 423
were harvested and resuspended by PBS. HSV-1 was collected after three times of freezing and 424
thawing in the infected cells and titrated by plaque assay in 12-well plates in Vero cells. 425
426
Plaque assay 427
Vero cells were cultured in a 12-well plate at a density of 2 × 105 cells per well, and infected 428
with 100 μl serially diluted HSV-1 supernatant for 2 h. Then, the cells were washed by PBS and 429
then immediately replenished with plaque medium supplemented with 1% carboxylmethylcelluose. 430
The infected Vero cells were incubated for 2–3 days. After the incubation, plaque medium was 431
removed and cells were fixed and stained with 4% formaldehyde and 0.5% crystal violet. 432
433
THP-1 macrophages stimulation 434
THP-1 cells were differentiated into macrophages with 100 ng/ml 435
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phorbol-12-myristate-13-acetate (TPA) for 12 h, and cells were cultured for 24 h without TPA. 436
Differentiated cells were then stimulated with HSV-1 infection, HSV120 transfection, DNA90 437
transfection, Sendai virus (SeV) infection, Zika virus (ZIKV) infection, or Nigericin treatment. 438
Supernatants were collected for measurement of IL-1β by Enzyme-linked immunosorbent assay 439
(ELISA). Cells were harvested for real-time PCR or immunoblot analysis. 440
441
Plasmid construction 442
The cDNAs encoding human STING, NLRP3, ASC, pro-Casp-1, and IL-1β were obtained by 443
reverse transcription of total RNA from TPA-differentiated THP-1 cells, followed by PCR using 444
specific primers. The cDNAs were sub-cloned into pcDNA3.1(+) and pcagg-HA vector. The 445
pcDNA3.1(+)-3×Flag vector was constructed from pcDNA3.1(+) vector through inserting the 446
3×Flag sequence between the NheI and HindIII site. Following are the primers used in this study. 447
Flag-NLRP3: 5’-CGCGGATCCATGAAGATGGCAAGCACCCGC-3’, 448
5’-CCGCTCGAGCTACCAAGAAGGCTCAAAGAC-3’; Flag-ASC: 449
5’-CCGGAATTCATGGGGCGCGCGCGCGACGCCAT-3’, 450
5’-CCGCTCGAGTCAGCTCCGCTCCAGGTCCTCCA-3’; Flag-Casp-1: 451
5’-CGCGGATCCATGGCCGACAAGGTCCTGAAG-3’, 452
5’-CCGCTCGAGTTAATGTCCTGGGAAGAGGTA-3’; Flag-IL-1β: 453
5’-CCGGAATTCATGGCAGAAGTACCTGAGCTC-3’, 454
5’-CCGCTCGAGTTAGGAAGACACAAATTGCAT-3’; Flag-STING: 455
5’-CCGGAATTCTATGCCCCACTCCAGCCTGCA-3’, 456
5'-CCGCTCGAGTCAAGAGAAATCCGTGCGGAG-3’. HA-NLRP3: 457
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5’-TACGAGCTCATGAAGATGGCAAGCACCCGC-3’, 458
5’-CCGCTCGAGCCAAGAAGGCTCAAAGACGAC-3’; HA-ASC: 459
5’-CCGGAATTCATGGGGCGCGCGCGCGACGCC-3’, 460
5’-CCGCTCGAGGCTCCGCTCCAGGTCCTCCAC-3’; HA-Casp-1: 461
5’-CCGGAATTCATGGCCGACAAGGTCCTGAAG-3’, 462
5’-CCGCTCGAGATGTCCTGGGAAGAGGTAGAA-3’. 463
The STING truncates was cloned into pcDNA3.1(+) and the PYRIN, NACHT, and LRR 464
domain of NLRP3 protein was cloned into pcDNA3.1(+) and pcaggs-HA vector using specific 465
primers, which are listed as follows. Flag-STING(1-160): 466
5’-CGCGGATCCATGCCCCACTCCAGCCTGCAT-3’, 467
5’-CCGGAATTCTGCCAGCCCATGGGCCACGTT-3’; Flag-STING(1-240): 468
5’-CGCGGATCCATGCCCCACTCCAGCCTGCAT-3’, 469
5’-CCGGAATTCGTAAACCCGATCCTTGATGCC-3; Flag-STING(41-379): 470
5’-CGCGGATCCATGGAGCACACTCTCCGGTAC-3’, 471
5’-CCGGAATTCTCAAGAGAAATCCGTGCGGAG-3’; Flag-STING(81-379): 472
5’-CGCGGATCCATGTACTGGAGGACTGTGCGG-3’, 473
5’-CCGGAATTCTCAAGAGAAATCCGTGCGGAG-3’; Flag-STING(111-379): 474
5’-CGCGGATCCATGGCGGTCGGCCCGCCCTTC-3’, 475
5’-CCGGAATTCTCAAGAGAAATCCGTGCGGAG-3’; Flag-STING(151-379): 476
5’-CGCGGATCCATGAATTTCAACGTGGCCCAT-3’, 477
5’-CCGGAATTCTCAAGAGAAATCCGTGCGGAG-3’, Flag-STING(211-379): 478
5’-CGCGGATCCATGCTGGATAAACTGCCCCAG-3’, 479
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5’-CCGGAATTCTCAAGAGAAATCCGTGCGGAG-3’; HA-PYRIN: 480
5’-CCGGAATTCATGAAGATGGCAAGCACCCGC-3’, 481
5’-CCGCTCGAGTAAACCCATCCACTCCTCTTC-3’; HA-NACHT: 482
5’-CCGGAATTCATGCTGGAGTACCTTTCGAGA-3’, HA-LRR: 483
5’-ATCGAGCTCATGTCTCAGCAAATCAGGCTG-3’, 484
5’-CCGCTCGAGCCAAGAAGGCTCAAAGACGAC-3’. 485
486
Lentivirus production and infection. 487
The targeting sequences of shRNAs for the human STING, NLRP3, ASC and Casp-1 were as 488
follows: sh-STING: GCCCGGATTCGAACTTACAAT; sh-NLRP3: 489
5’-CAGGTTTGACTATCTGTTCT-3’; sh-ASC: 5’-GATGCGGAAGCTCTTCAGTTTCA-3’; 490
sh-caspase-1: 5’-GTGAAGAGATCCTTCTGTA-3’. A PLKO.1 vector encoding shRNA for a 491
negative control (Sigma-Aldrich, St. Louis, MO, USA) or a specific target molecule (Sigma-Aldrich) 492
was transfected into HEK293T cells together with psPAX2 and pMD2.G with Lipofectamine 2000. 493
Culture supernatants were harvested 36 and 60 h after transfection and then centrifuged at 2,200 494
rpm for 15 min. THP-1 cells were infected with the supernatants contain lentiviral particles in the 495
presence of 4 μg/ml polybrene (Sigma). After 48 h of culture, cells were selected by 1.5 μg/ml 496
puromycin (Sigma). Hela cells were selected by 2.5 μg/ml puromycin (Sigma). The results of each 497
sh-RNA-targeted protein were detected by immunoblot analysis. 498
499
Enzyme-linked immunosorbent assay (ELISA) 500
Concentrations of human IL-1β in culture supernatants were measured by ELISA kit (BD 501
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Biosciences, San Jose, CA, USA). The mouse IL-1β ELISA Kit was purchased from R&D. 502
503
Activated caspase-1 and mature IL-1β measurement 504
Supernatant of the cultured cells was collected for 1 ml in the cryogenic vials (Corning). The 505
supernatant was centrifuged at 12,000 rpm for 10 min each time by Amicon Ultra (UFC500308) 506
from Millipore for protein concentrate. The concentrated supernatant was mixed with SDS loading 507
buffer for western blotting analysis with antibodies for detection of activated caspase-1 (D5782 508
1:500; Cell Signaling) or mature IL-1β (Asp116 1:500; Cell Signaling). Adherent cells in each well 509
were lysed with the lyses buffer described below, followed by immunoblot analysis to determine the 510
cellular content of various protein. 511
512
Western blot analysis 513
HEK293T whole-cell lysates were prepared by lysing cells with buffer (50 mM Tris-HCl, 514
pH7.5, 300 mM NaCl, 1% Triton-X, 5 mM EDTA and 10% glycerol). The TPA-differentiated 515
THP-1 cells lysates were prepared by lysing cells with buffer (50 mM Tris-HCl, pH7.5, 150 mM 516
NaCl, 0.1% Nonidetp 40, 5 mM EDTA and 10% glycerol). Protein concentration was determined by 517
Bradford assay (Bio-Rad, Hercules, CA, USA). Cultured cell lysates (30 μg) were electrophoresed 518
in an 8-12% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, MA, USA). PVDF 519
membranes were blocked with 5% skim milk in phosphate buffered saline with 0.1% Tween 20 520
(PBST) before being incubated with the antibody. Protein band were detected using a Luminescent 521
image Analyzer (Fujifilm LAS-4000). 522
523
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Co-immunoprecipitation assays 524
HEK293T whole-cell lysates were prepared by lysing cells with buffer (50 mM Tris-HCl, 525
pH7.5, 300 mM NaCl, 1% Triton-X, 5 mM EDTA, and 10% glycerol). TPA-differentiated THP-1 526
cells lysates were prepared by lysing cells with buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% 527
Nonidetp40, 5 mM EDTA, and 10% glycerol). Lysates were immunoprecipitated with control 528
mouse immunoglobulin G (IgG) (Invitrogen) or anti-Flag antibody (Sigma, F3165) with Protein-G 529
Sepharose (GE Healthcare, Milwaukee, WI, USA). 530
531
Confocal microscopy 532
HEK293T cells and Hela cells were transfected with plasmids for 24–36 h. Cells were fixed 533
in 4% paraformaldehyde at room temperature for 15 min. After being washed three times with PBS, 534
permeabilized with PBS containing 0.1% Triton X-100 for 5 min, washed three times with PBS, and 535
finally blocked with PBS containing 5% BSA for 1 h. The cells were then incubated with the 536
monoclonal mouse anti-Flag antibody (F3165, Sigma) and Monoclonal rabbit anti-HA (H6908, 537
Sigma) overnight at 4°C, followed by incubation with FITC-conjugate donkey anti-mouse IgG 538
(Abbkine) and Dylight 649-conjugate donkey anti-rabbit IgG (Abbkine) for 1 h. After washing three 539
times, cells were incubated with DAPI solution for 5 min, and then washed three more times with 540
PBS. Finally, the cells were analyzed using a confocal laser scanning microscope (Fluo View 541
FV1000; Olympus, Tokyo, Japan). 542
543
Real-time PCR 544
Total RNA was extracted with TRIzol reagent (Invitrogen), following the manufacturer’s 545
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instructions. Real-time quantitative-PCR was performed using the Roche LC480 and SYBR 546
qRT-PCR kits (DBI Bio-science, Ludwigshafen, Germany) in a reaction mixture of 20 μl SYBR 547
Green PCR master mix, 1 μl DNA diluted template, and RNase-free water to complete the 20 μl 548
volume. Real-time PCR primers were designed by Primer Premier 5.0 and their sequences were as 549
follows: HSV-1 ICP27 forward, 5’-GCATCCTTCGTGTTTGTCATT-3’, HSV-1 ICP27 reverse, 550
5’-GCATCTTCTCTCCGACCCCG-3’. HSV-1 UL30 forward, 551
5’-CATCACCGACCCGGAGAGGGAC-3’, HSV-1 UL30 reverse, 552
5’-GGGCCAGGCGCTTGTTGGTGTA-3’. SeV P protein forward, 5’- 553
CAAAAGTGAGGGCGAAGGAGAA-3’, SeV P protein reverse, 5’- 554
CGCCCAGATCCTGAGATACAGA-3’. ZIKV forward, 555
5’-GGTCAGCGTCCTCTCTAATAAACG-3’, ZIKV reverse, 556
5’-GCACCCTAGTGTCCACTTTTTCC-3’. IL-1β forward, 557
5’-CACGATGCACCTGTACGATCA-3’, IL-1β reverse, 5’-GTTGCTCCATATCCTGTCCCT-3’. 558
GAPDH forward, 5’-AAGGCTGTGGGCAAGG-3’, GAPDH reverse, 559
5’-TGGAGGAGTGGGTGTCG-3’. Mouse GAPDH forward, 5’-TGGCCTTCCGTGTTCCTAC-3’, 560
Mouse GAPDH reverse, 5’-GAGTTGCTGTTGAAGTCGCA-3’. Mouse IL-1β forward, 561
5’-GAAATGCCACCTTTTGACAGTG-3’, Mouse IL-1β reverse, 562
5’-TGGATGCTCTCATCAGGACAG-3’. Mouse IL-6 forward, 563
5’-ACAAAGCCAGAGTCCTTCAGA -3’, Mice IL-6 reverse, 564
5’-TCCTTAGCCACTCCTTCTGT-3’. Mice TNF-α forward, 565
5’-ACTGAACTTCGGGGTGATCG-3’, Mice TNF-α reverse, 566
5’-TCTTTGAGATCCATGCCGTTG-3’. 567
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568
ASC oligomerization detection 569
HEK293T cells were transfected with plasmids for 24–36 h. Cell lysates were centrifugated at 570
6000 rpm for 15 min. The supernatants of the lysates were mixed with SDS loading buffer for 571
western blot analysis with antibody against ASC. The pellets of the lysates were washed with PBS 572
for three times and cross-linked using fresh DSS (2 mM, Sigma) at 37°C for 30 min. The 573
cross-linked pellets were then spanned down and the supernatant was mixed with SDS loading 574
buffer for western blotting analysis. 575
576
Statistical analyses 577
All experiments were reproducible and repeated at least three times with similar results. 578
Parallel samples were analyzed for normal distribution using Kolmogorov-Smirnov tests. Abnormal 579
values were eliminated using a follow-up Grubbs test. Levene’s test for equality of variances was 580
performed, which provided information for Student’s t-tests to distinguish the equality of means. 581
Means were illustrated using histograms with error bars representing the SD; a P value of <0.05 was 582
considered statistically significant. 583
584
585
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29
Acknowledgments 586
We thank Dr. Di Wang of Zhejiang University School of Medicine, China for kindly providing 587
C57BL/6 NLRP3-/- mice, Dr. Jun Cui of Sun Yat-Sen University, Guangzhou 510275, China for 588
Human acute monocytic leukemia (THP-1) cells, and Dr. Bo Zhong of Wuhan University, China, 589
for kindly providing Herpes simplex virus 1 strain, Sendai virus strain, and plasmids encoding 590
ubiquitin and mutants. 591
This work was supported by National Natural Science Foundation of China (81730061 and 592
81471942), Postdoctoral Science Foundation of China (2018T110923), National Health and Family 593
Planning Commission of China (National Mega Project on Major Infectious Disease Prevention) 594
(2017ZX10103005 and 2017ZX10202201), and Guangdong Province “Pearl River Talent Plan” 595
Innovation and Entrepreneurship Team Project (2017ZT07Y580). 596
597
Author contributions: 598
W.W., D.H., Y.F., Q.Z., W.L., F.L., K.W., G.L., Y.L., and J.W. contributed the conceptualization. 599
W.W., D.H., Y.F., C.W., A.L., Y.W., K.C., M.T., F.X., Q.Z., M.A.S., W.C., P.P., P.W., and W.L. 600
contributed the methodology, conduction of experiments, and investigation. W.W., F.L., K.W., G.L., 601
Y.L., and J.W. contributed the reagents and resources. W.W., and J.W. contributed the writing the 602
original draft of the paper. W.W., and J.W. contributed the review and editing the paper. F.L., K.W., 603
G.L., and Y.L. contributed the visualization or funding acquisition. J.W. contributed the supervision 604
and funding acquisition. 605
Conflict of Interests: The authors have no conflict of interests. 606
607
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30
References 608
609
Abe T, Barber GN (2014) Cytosolic-DNA-mediated, STING-dependent proinflammatory gene 610
induction necessitates canonical NF-kappaB activation through TBK1. J Virol 88: 5328-5341 611
Abe T, Harashima A, Xia T, Konno H, Konno K, Morales A, Ahn J, Gutman D, Barber GN (2013) 612
STING recognition of cytoplasmic DNA instigates cellular defense. Mol Cell 50: 5-15 613
Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124: 614
783-801 615
Barry R, John SW, Liccardi G, Tenev T, Jaco I, Chen CH, Choi J, Kasperkiewicz P, 616
Fernandes-Alnemri T, Alnemri E, Drag M, Chen Y, Meier P (2018) SUMO-mediated regulation 617
of NLRP3 modulates inflammasome activity. Nat Commun 9: 3001 618
Chen J, Chen ZJ (2018) PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome 619
activation. Nature 564: 71-76 620
Chen Q, Sun L, Chen ZJ (2016) Regulation and function of the cGAS-STING pathway of cytosolic 621
DNA sensing. Nat Immunol 17: 1142-1149 622
Compan V, Baroja-Mazo A, Lopez-Castejon G, Gomez AI, Martinez CM, Angosto D, Montero MT, 623
Herranz AS, Bazan E, Reimers D, Mulero V, Pelegrin P (2012) Cell volume regulation 624
modulates NLRP3 inflammasome activation. Immunity 37: 487-500 625
Dinarello CA (2009) Immunological and inflammatory functions of the interleukin-1 family. Annu 626
Rev Immunol 27: 519-550 627
Gaidt MM, Ebert TS, Chauhan D, Ramshorn K, Pinci F, Zuber S, O'Duill F, Schmid-Burgk JL, Hoss 628
F, Buhmann R, Wittmann G, Latz E, Subklewe M, Hornung V (2017) The DNA inflammasome 629
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
31
in human myeloid cells is initiated by a STING-cell death program upstream of NLRP3. Cell 630
171: 1110-1124 631
Guo C, Chi Z, Jiang D, Xu T, Yu W, Wang Z, Chen S, Zhang L, Liu Q, Guo X, Zhang X, Li W, Lu 632
L, Wu Y, Song BL, Wang D (2018) Cholesterol homeostatic regulator SCAP-SREBP2 633
integrates NLRP3 inflammasome activation and cholesterol biosynthetic signaling in 634
macrophages. Immunity 49: 842-856 635
Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, 636
Moore KJ, Golenbock DT (2008) The NALP3 inflammasome is involved in the innate immune 637
response to amyloid-beta. Nat Immunol 9: 857-865 638
Hardison SE, Brown GD (2012) C-type lectin receptors orchestrate antifungal immunity. Nat 639
Immunol 13: 817-822 640
Hottz ED, Lopes JF, Freitas C, Valls-de-Souza R, Oliveira MF, Bozza MT, Da PA, Weyrich AS, 641
Zimmerman GA, Bozza FA, Bozza PT (2013) Platelets mediate increased endothelium 642
permeability in dengue through NLRP3-inflammasome activation. Blood 122: 3405-3414 643
Humphries F, Bergin R, Jackson R, Delagic N, Wang B, Yang S, Dubois AV, Ingram RJ, Moynagh 644
PN (2018) The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome. Nat 645
Commun 9: 1560 646
Ishikawa H, Barber GN (2008) STING is an endoplasmic reticulum adaptor that facilitates innate 647
immune signalling. Nature 455: 674-678 648
Ishikawa H, Ma Z, Barber GN (2009) STING regulates intracellular DNA-mediated, type I 649
interferon-dependent innate immunity. Nature 461: 788-792 650
Johnson KE, Chikoti L, Chandran B (2013) Herpes simplex virus 1 infection induces activation and 651
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
32
subsequent inhibition of the IFI16 and NLRP3 inflammasomes. J Virol 87: 5005-5018 652
Juliana C, Fernandes-Alnemri T, Kang S, Farias A, Qin F, Alnemri ES (2012) Non-transcriptional 653
priming and deubiquitination regulate NLRP3 inflammasome activation. J Biol Chem 287: 654
36617-36622 655
Kanneganti TD, Body-Malapel M, Amer A, Park JH, Whitfield J, Franchi L, Taraporewala ZF, 656
Miller D, Patton JT, Inohara N, Nunez G (2006) Critical role for Cryopyrin/Nalp3 in activation 657
of caspase-1 in response to viral infection and double-stranded RNA. J Biol Chem 281: 658
36560-36568 659
Komune N, Ichinohe T, Ito M, Yanagi Y (2011) Measles virus V protein inhibits NLRP3 660
inflammasome-mediated interleukin-1beta secretion. J Virol 85: 13019-13026 661
Lawrence TM, Hudacek AW, de Zoete MR, Flavell RA, Schnell MJ (2013) Rabies virus is 662
recognized by the NLRP3 inflammasome and activates interleukin-1beta release in murine 663
dendritic cells. J Virol 87: 5848-5857 664
Martinon F, Agostini L, Meylan E, Tschopp J (2004) Identification of bacterial muramyl dipeptide as 665
activator of the NALP3/cryopyrin inflammasome. Curr Biol 14: 1929-1934 666
Martinon F, Mayor A, Tschopp J (2009) The inflammasomes: guardians of the body. Annu Rev 667
Immunol 27: 229-265 668
Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J (2006) Gout-associated uric acid crystals 669
activate the NALP3 inflammasome. Nature 440: 237-241 670
Maruzuru Y, Ichinohe T, Sato R, Miyake K, Okano T, Suzuki T, Koshiba T, Koyanagi N, Tsuda S, 671
Watanabe M, Arii J, Kato A, Kawaguchi Y (2018) Herpes simplex virus 1 VP22 inhibits 672
AIM2-dependent inflammasome activation to enable efficient viral replication. Cell Host 673
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
33
Microbe 23: 254-265 674
Muruve DA, Petrilli V, Zaiss AK, White LR, Clark SA, Ross PJ, Parks RJ, Tschopp J (2008) The 675
inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune 676
response. Nature 452: 103-107 677
Negash AA, Ramos HJ, Crochet N, Lau DT, Doehle B, Papic N, Delker DA, Jo J, Bertoletti A, 678
Hagedorn CH, Gale MJ (2013) IL-1beta production through the NLRP3 inflammasome by 679
hepatic macrophages links hepatitis C virus infection with liver inflammation and disease. PLoS 680
Pathog 9: e1003330 681
Py BF, Kim MS, Vakifahmetoglu-Norberg H, Yuan J (2013) Deubiquitination of NLRP3 by BRCC3 682
critically regulates inflammasome activity. Mol Cell 49: 331-338 683
Rajan JV, Rodriguez D, Miao EA, Aderem A (2011) The NLRP3 inflammasome detects 684
encephalomyocarditis virus and vesicular stomatitis virus infection. J Virol 85: 4167-4172 685
Ramos HJ, Lanteri MC, Blahnik G, Negash A, Suthar MS, Brassil MM, Sodhi K, Treuting PM, 686
Busch MP, Norris PJ, Gale MJ (2012) IL-1beta signaling promotes CNS-intrinsic immune 687
control of West Nile virus infection. PLoS Pathog 8: e1003039 688
Roizman, B., Knipe, D.M., and Whitley, R.J. (2013) Herpes simplex viruses. In Fields Virology. 689
Knipe, D. M. (ed) pp 1823–1897. Lippincott-Williams &Wilkins Press. 690
Schroder K, Tschopp J (2010) The inflammasomes. Cell 140: 821-832 691
Schroder K, Zhou R, Tschopp J (2010) The NLRP3 inflammasome: a sensor for metabolic danger? 692
Science 327: 296-300 693
Shenoy AR, Wellington DA, Kumar P, Kassa H, Booth CJ, Cresswell P, MacMicking JD (2012) 694
GBP5 promotes NLRP3 inflammasome assembly and immunity in mammals. Science 336: 695
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
34
481-485 696
Song H, Liu B, Huai W, Yu Z, Wang W, Zhao J, Han L, Jiang G, Zhang L, Gao C, Zhao W (2016) 697
The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation by promoting 698
proteasomal degradation of NLRP3. Nat Commun 7: 13727 699
Song N, Li T (2018) Regulation of NLRP3 Inflammasome by Phosphorylation. Front Immunol 9: 700
2305 701
Subramanian N, Natarajan K, Clatworthy MR, Wang Z, Germain RN (2013) The adaptor MAVS 702
promotes NLRP3 mitochondrial localization and inflammasome activation. Cell 153: 348-361 703
Swanson KV, Junkins RD, Kurkjian CJ, Holley-Guthrie E, Pendse AA, El MR, Petrucelli A, Barber 704
GN, Benedict CA, Ting JP (2017) A noncanonical function of cGAMP in inflammasome 705
priming and activation. J Exp Med 214: 3611-3626 706
Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140: 805-820 707
Ting JP, Lovering RC, Alnemri ES, Bertin J, Boss JM, Davis BK, Flavell RA, Girardin SE, Godzik 708
A, Harton JA, Hoffman HM, Hugot JP, Inohara N, Mackenzie A, Maltais LJ, Nunez G, Ogura 709
Y, Otten LA, Philpott D, Reed JCet al (2008) The NLR gene family: a standard nomenclature. 710
Immunity 28: 285-287 711
Wang H, Lei X, Xiao X, Yang C, Lu W, Huang Z, Leng Q, Jin Q, He B, Meng G, Wang J (2015) 712
Reciprocal Regulation between Enterovirus 71 and the NLRP3 Inflammasome. Cell Rep 12: 713
42-48 714
Wang W, Li G, De Wu, Luo Z, Pan P, Tian M, Wang Y, Xiao F, Li A, Wu K, Liu X, Rao L, Liu F, 715
Liu Y, Wu J (2018) Zika virus infection induces host inflammatory responses by facilitating 716
NLRP3 inflammasome assembly and interleukin-1beta secretion. Nat Commun 9: 106 717
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
35
Wang W, Xiao F, Wan P, Pan P, Zhang Y, Liu F, Wu K, Liu Y, Wu J (2017) EV71 3D Protein binds 718
with NLRP3 and enhances the assembly of inflammasome complex. PLoS Pathog 13: 719
e1006123 720
Webster SJ, Brode S, Ellis L, Fitzmaurice TJ, Elder MJ, Gekara NO, Tourlomousis P, Bryant C, 721
Clare S, Chee R, Gaston H, Goodall JC (2017) Detection of a microbial metabolite by STING 722
regulates inflammasome activation in response to Chlamydia trachomatis infection. PLoS 723
Pathog 13: e1006383 724
Wu J, Sun L, Chen X, Du F, Shi H, Chen C, Chen ZJ (2013) Cyclic GMP-AMP is an endogenous 725
second messenger in innate immune signaling by cytosolic DNA. Science 339: 826-830 726
Yan Y, Jiang W, Liu L, Wang X, Ding C, Tian Z, Zhou R (2015) Dopamine controls systemic 727
inflammation through inhibition of NLRP3 inflammasome. Cell 160: 62-73 728
Ye Z, Ting JP (2008) NLR, the nucleotide-binding domain leucine-rich repeat containing gene 729
family. Curr Opin Immunol 20: 3-9 730
Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, 731
Fujita T (2004) The RNA helicase RIG-I has an essential function in double-stranded 732
RNA-induced innate antiviral responses. Nat Immunol 5: 730-737 733
734
735
736
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36
Figure Legends 737
738
Figure 1 – STING interacts with NLRP3 to facilitate the inflammasome activation. 739
A–C HEK293T cells were co-transfected with pFlag-STING and pHA-NLRP3 (A and B), or with 740
pFlag-STING, pHA-NLRP3, pHA-ASC, and pHA-Casp-1 (C). 741
D Diagrams of NLRP3, PYRIN, NACHT, and LRR. 742
E, F HEK293T cells were co-transfected with pHA-STING and pFlag-NLRP3, pFlag-PYRIN, 743
pFlag-NACHT, or pFlag-LRR (E), or with pFlag-STING and pHA-NLRP3, pHA-PYRIN, 744
pHA-NACHT, or pHA-LRR (F). 745
G Diagrams of STING and its truncated proteins. 746
H HEK293T cells were co-transfected with pHA-NLRP3 and pFlag-STING or truncated proteins. 747
I HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Casp-1, and 748
pro-IL-1β to generate a pro-IL-1β cleavage system (NACI), and transfected with pFlag-STING or 749
truncated proteins. IL-1β in supernatants was determined by ELISA. 750
J HEK293T cells were co-transfected with pFlag-ASC, pHA-NLRP3, or pFlag-STING. 751
A–C, E, F, H and J Cell lysates were subjected to co-immunoprecipitation (Co-IP) using IgG 752
(Mouse) and anti-Flag antibody (A, F), IgG (Rabbit) and anti-HA antibody (B, J), anti-HA antibody 753
(C), and anti-Flag antibody (E, H), and analyzed by immunoblotting using anti-HA or anti-Flag 754
antibody (top) or subjected directly to Western blot using anti-Flag or anti-HA antibody (input) 755
(bottom). 756
K HEK293T cells were co-transfected with pFlag-ASC, pHA-NLRP3, and pMyc-STING. Pellets 757
were subjected to ASC oligomerization assays (top) and lysates were prepared for Western blots 758
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
37
(bottom). 759
L, M HEK293T cells (L) or Hela cells (M) were co-transfected with pFlag-STING and/or 760
pHA-NLRP3. Sub-cellular localization of Flag-STING (green), HA-NLRP3 (red), and DAPI (blue) 761
were examined by confocal microscopy. 762
Data shown are means ± SEMs; **p < 0.01, ***p < 0.0001; ns, no significance. 763
764
Figure 2 – HSV-1 infection promotes the STING-NLRP3 interaction. 765
A, B TPA-differentiated THP-1 macrophages were mock-infected or infected with HSV-1 (MOI=1) 766
for 4 h. 767
C, D HEK293T cells (C) and Hela cells (D) were co-transfected with pFlag-STING and 768
pHA-NLRP3, and then infected with HSV-1 (MOI=1) for 2–4 h. 769
E TPA-differentiated THP-1 macrophages were transfected with HSV120 (3 µg/ml) by Lipo2000 770
for 4 h. 771
F, G HEK293T cells (F) and Hela cells (G) were co-transfected with pFlag-STING and 772
pHA-NLRP3 and then transfected with HSV120 (3 µg/ml) for 4 h. 773
H Primary MEFs were primed with LPS (1 µg/ml) for 6 h, and then infected with HSV-1 (MOI=1) 774
for 4 h or transfected with HSV120 (3 µg/ml) for 4 h. 775
A–H Cell lysates were subjected to Co-IP using IgG (Mouse) or anti-NLRP3 antibody (A), 776
anti-STING antibody (B), IgG (Mouse) or anti-Flag antibody (C), anti-Flag antibody (D), IgG 777
(Mouse) or anti-NLRP3 antibody (E), IgG (Mouse) or anti-Flag antibody (F), anti-Flag antibody (G), 778
and IgG (Mouse) or anti-NLRP3 antibody (H), and then analyzed by immunoblotting using 779
anti-NLRP3 and anti-STING antibody (top) or analyzed directly by immunoblotting using 780
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anti-NLRP3 and anti-STING antibody (as input) (bottom). 781
782
Figure 3 – HSV-1 infection induces IL-1β expression and secretion. 783
A–K TPA-differentiated THP-1 macrophages were treated with 2 μM Nigericin for 2 h, and infected 784
with HSV-1 at MOI=0.1, 0.2, 0.4, and 0.8 for 8 h (A, C, E and G), infected with HSV-1 at MOI=0.8 785
for 2, 4, 6, and 8 h (B, D, F and H), or transfected with HSV120 (3 µg/ml) for 2, 4, and 6 h (I–K). 786
L–O Mice primary MEFs were primed with LPS (1 µg/ml) for 6 h, and then treated with 5 mM ATP 787
for 2 h or infected with HSV-1 (MOI=1) (L and N) or transfected with HSV120 (3 µg/ml) (M, O) 788
for 2, 4, and 6 h. 789
A, B, I, L and M IL-1β protein was determined by ELISA. 790
C, D and J Matured IL-1β (p17) and cleaved Casp-1 in supernatants (top) and pro-IL-1β production 791
in lysates (bottom) were determined by Western-blot analyses. 792
E, F, G, H, K, N and O IL-1β mRNA and GAPDH mRNA (E, F), HSV-1 ICP27 mRNA and 793
GAPDH mRNA (G, H and N), and IFN-β mRNA and GAPDH mRNA (K, O) were quantified by 794
RT-PCR. 795
Data shown are means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.0001; ns, no significance. 796
797
Figure 4 – The NLRP3 inflammasome is required for HSV-1-induced IL-1β activation. 798
A–F TPA-differentiated THP-1 macrophages were treated with 2 μM Nigericin for 2 h, infected 799
with HSV-1 at MOI=0.8 for 8 h, and then treated with Glybenclamide (25 µg/ml) (A, B and E) or 800
Ac-YVAD-cmk (10 µg/ml) (C, D and F). 801
G–K THP-1 cells stably expressing shRNAs targeting NLRP3, ASC or Casp-1 were generated and 802
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39
then treated with 2 µM Nigericin for 2 h (G, H) or infected with HSV-1 at MOI=0.8 for 8 h (I–K). 803
L–N Primary MEFs of C57BL/6 WT mice and C57BL/6 NLRP3-/- mice were primed with LPS (1 804
µg/ml) for 6 h, and then treated with 5 mM ATP for 2 h or infected with HSV-1 (MOI=1) for 6 h. 805
NLRP3 and GAPDH protein in lysates were determined by Western blot (L). IL-1β levels were 806
determined by ELISA (A, C, G and M). Matured IL-1β (p17) and cleaved Casp-1 (p22/p20) in 807
supernatants (top) or pro-IL-1β and pro-Casp-1 in lysates (bottom) were determined by Western-blot 808
(B, H and J). HSV-1 ICP27 mRNA and GAPDH mRNA were quantified by RT-PCR (E, F, K and N). 809
Data shown are means ± SEMs; ***p < 0.0001; ns, no significance. 810
811
Figure 5 – STING recruits NLRP3 to the ER and promotes the inflammasome formation. 812
A–D Hela cells were co-transfected with pFlag-STING and pHA-NLRP3 (A), transfected with 813
pFlag-NLRP3 and infected with HSV-1 (MOI=1) for 4 h or transfected with HSV120 (3 µg/ml) for 814
4 h (B, C and D). Sub-cellular localization of HA-NLRP3 (green) (A), Flag-STING (red) (A), ER 815
Blue (blue) (A, D), Flag-NLRP3 (red) (B, C), Calnexin (green) (B), DAPI (blue) (B and C), 816
Calnexin (green) (C) and HA-NLRP3 (red) (D) were examined by confocal microscopy. 817
E–G Hela cells were co-transfected with pFlag-STING and pHA-NLRP3 (E). THP-1 macrophages 818
were infected with HSV-1 (MOI=1) for 4 h (F). Mice primary MEFs were primed with LPS (1 819
µg/ml) for 6 h and infected with HSV-1 (MOI=1) for 4 h (G). Flag-NLRP3, HA-STING, Calnexin, 820
and GAPDH in whole cell lysate (WCL) and purified ER were determined by Western-blot 821
analyses. 822
H, I Hela cells stably expressing sh-STNG were transfected with pFlag-NLRP3 and infected with 823
HSV-1 (MOI=1) for 4 h or transfected with HSV120 (3 µg/ml) for 4 h. Sub-cellular localization of 824
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40
Flag-NLRP3 (red) (H), Calnexin (green) (H), DAPI (blue) (H), HA-NLRP3 (red) (I) and ER blue (I) 825
were examined by confocal microscopy. 826
J–L Hela cells stably expressing sh-STING were transfected with pHA-NLRP3 and infected with 827
HSV-1 (MOI=1) for 4 h (J). THP-1 cells stably expressing sh-STING were differentiated to 828
macrophages, and infected with HSV-1 (MOI=1) for 4 h (K). Primary MEFs stably expressing 829
sh-STING were primed with LPS (1 µg/ml) for 6 h, and infected with HSV-1 (MOI=1) for 4 h (L). 830
HA-NLRP3 (J), NLRP3 (K, I), STING (J–L), Calnexin (ER) (J–L), and GAPDH (J–L) in WCL and 831
purified ER fraction were determined by Western-blot analyses. 832
833
Figure 6 – STING deubiquitinates NLRP3 to activate the NLRP3 inflammasome. 834
A–E Hela cells were co-transfected with pFlag-NLRP3, pHA-Ubiquitin, and pMyc-STING (A), 835
with pFlag-NLRP3, pHA-Ubiquitin, pHA-Ubiquitin mutations (K48R), pHA-Ubiquitin mutations 836
(K63R) and pMyc-STING (B), with pFlag-NLRP3, pHA-Ubiquitin, pHA-Ubiquitin 837
mutations(K48O), pHA-Ubiquitin mutations(K63O), and pMyc-STING (C), with pFlag-NLRP3 and 838
pHA-Ubiquitin and then infected with HSV-1(MOI=1) for 2 and 4 h (D), and transfected with 839
HSV120 (3 µg/ml) for 2 and 4 h (E). Cell lysates were prepared and subjected to denature-IP using 840
anti-Flag antibody and then analyzed by immunoblotting using an anti-HA or anti-Flag antibody 841
(top) or subjected directly to Western blot using an anti-Flag, anti-HA or anti-Myc antibody (as 842
input) (bottom). 843
F–I TPA-differentiated THP-1 macrophages were infected with HSV-1 (MOI=1) for 2 and 4 h (F), 844
and transfected with HSV120 (3 µg/ml) for 2, 4 and 6 h (G). Mice primary MEFs were infected 845
with HSV-1 (MOI=1) for 2 h (H). THP-1 cells stably expressing sh-NC or sh-STING were 846
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
41
generated and differentiated to macrophages and then infected with HSV-1 (MOI=1) for 4 h (I). Cell 847
lysates were prepared and subjected to denature-IP using anti-NLRP3 antibody and then analyzed 848
by immunoblotting using an anti-Ubiquitin, anti-K48-Ubiquitin, anti-K63-Ubiquitin or anti-NLRP3 849
antibody (top) (F) or using anti-NLRP3 antibody (G, H and I), or subjected directly to Western blot 850
using an anti- Ubiquitin or anti-NLRP3 antibody (as input) (bottom). 851
852
Figure 7 – STING is required for the NLRP3 inflammasome activation upon DNA virus 853
infection. 854
A–H THP-1 cells stably expressing sh-NC or sh-STING were generated and differentiated to 855
macrophages, and then infected with HSV-1 at MOI=0.8 for 2, 4, and 8 h (A–C), transfected with 856
DNA90 (3 µg/ml), HSV120 (3 µg/ml) or infected with HSV-1 at MOI=0.8 for 8 h (D, E), and 857
treated with 2 μM Nigericin for 2 h and infected with HSV-1 at MOI=0.8 for 8 h (F–H). IL-1β 858
levels were determined by ELISA (A, D and F). Matured IL-1β (p17) and cleaved Casp-1 (p22/p20) 859
in supernatants or STING, pro-IL-1β and pro-Casp-1 in lysates were determined by Western-blot (B, 860
C and G). HSV-1 ICP27 mRNA and GAPDH mRNA were quantified by RT-PCR (C, H). 861
I–M THP-1 cells stably expressing shRNA targeting STING was generated and differentiated to 862
macrophages, then infected with SeV (MOI=1) for 24 h, ZIKV (MOI=1) for 24 h or HSV-1 863
(MOI=0.8) for 8 h. IL-1β levels were determined by ELISA (I). Matured IL-1β (p17) and cleaved 864
Casp-1 (p22/p20) in supernatants or STING, pro-IL-1β and pro-Casp-1 in lysates were determined 865
by Western-blot (J). SeV P mRNA (K), ZIKV mRNA (L), HSV-1 ICP27 mRNA (M) and GAPDH 866
mRNA were quantified by RT-PCR. 867
Data shown are means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.0001; ns, no significance. 868
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
42
869
Figure 8 – NLRP3 is critical for host defense against HSV-1 infection in mice. 870
A, B C57BL/6 WT mice (8-week-old, female, n=9) and C57BL/6 NLRP3-/- mice (8-week-old, 871
female, n=5) were infected i.p. with HSV-1 at 1×107 pfu for 11 days. The survival rates (A) and 872
body weights (B) of mice were evaluated. 873
C–G WT mice (8-week-old, female) and NLRP3-/- mice (8-week-old, female) were mock-infected 874
i.p. with PBS (WT and NLRP3-/- mice, n=3) or infected i.p. with HSV-1 (WT mice, n=4, NLRP3-/- 875
mice, n=3) at 1×107 pfu for 6 h. IL-1β in mice sera was determined by ELISA (C). IL-1β mRNA (D), 876
IL-6 mRNA (E), TNF-α mRNA (F), HSV-1 UL30 mRNA (G) and GAPDH mRNA in mice blood 877
were quantified by RT-PCR. 878
H–J WT mice (8-week-old, female, n=4) and NLRP3-/- mice (8-week-old, female, n=4) were 879
infected i.p. with HSV-1 at 1×107 pfu for 1 days. IL-1β mRNA (H), IL-6 mRNA (I), and GAPDH 880
mRNA in mice lung were quantified by RT-PCR. HSV-1 viral titers were measured by plaque assays 881
for mice lung (J). 882
K–M WT mice (8-week-old, female, n=7) and NLRP3-/- mice (8-week-old, female, n=7) were 883
infected i.p. with HSV-1 at 1×107 pfu for 4 days. IL-1β mRNA (K), IL-6 mRNA (L) and GAPDH 884
mRNA in mice brain were quantified by RT-PCR. HSV-1 viral titers were measured by plaque 885
assays for mice brain (M). 886
N, O WT mice (8-week-old, female) and NLRP3-/- mice (8-week-old, female) were infected i.p. 887
with HSV-1 at 1×107 pfu for 1 day. The lung tissue (N) and brain tissue (O) were stained with 888
histological analysis (H&E or IL-1β). 889
Data shown are means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.0001; ns, no significance. 890
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
43
891
Fig. 9. A proposed model for the regulation of NLRP3 inflammasome activation mediated by 892
the cGAS-STING-NLRP3 pathway. 893
In resting cells and under normal physiological conditions, CUL1 interacts with NLRP3 to disrupt 894
the inflammasome assembly, and catalyzes NLRP3 ubiquitination to repress the inflammasome 895
activation (left). However, in response to inflammatory stimuli, CUL1 disassociates from NLRP3 to 896
release the repression of inflammasome assembly and activation (right). 897
898
899
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 24, 2019. ; https://doi.org/10.1101/681338doi: bioRxiv preprint